Microdroplet-manipulation systems and methods for automated execution of molecular biological protocols

Abstract

Disclosed herein are automated systems for performing various biochemical and molecular biological procedures, including processor-controlled execution of protocols involving multiple steps performed in, on, or with liquid microdroplets. Example protocols are the various Polymerase Chain Reaction (PCR) protocols, but the subject systems are not limited to performing PCR protocols. Formation of a microdroplet of the sample for use in the described systems is achieved by bringing an amount of the sample into contact with a hydrophobic milieu, such as a superhydrophobic surface or hydrophobic liquid.

What is claimed is: 
     
       1. An apparatus, comprising:
 a movement and placement device defining a preset motion range in at least two of x-, y-, and z-dimensions; 
 a microdroplet-manipulating device coupled to and movable by the controlled movement and placement device in the preset range; 
 a superhydrophobic surface located in the preset range; 
 a temperature-controlled vessel located in the preset range and comprising:
 multiple interconnected chambers each chamber comprising a respective volume of a hydrophobic liquid held at a respective temperature; and 
 a plurality of open-top channels interconnecting the chambers, each channel comprising a respective volume of the hydrophobic liquid; and 
 
 a controller operably connected to the movement and placement device and to the microdroplet-manipulating device, the controller programmed to:
 command the movement and positioning device to place the microdroplet-manipulating device relative to the superhydrophobic surface, and 
 command the movement and placement device to perform automatically at least one of:
 (a) placing an aqueous liquid microdroplet on the superhydrophobic surface, 
 (b) manipulating the microdroplet on the superhydrophobic surface, or 
 (c) removing at least a portion of the microdroplet from the superhydrophobic surface; and 
 
 at least one of:
 (d) placing the microdroplet in a first chamber of the temperature-controlled vessel, 
 (e) manipulating the microdroplet in the first chamber, or 
 (f) removing at least a portion of the microdroplet from the first chamber. 
 
 
 
     
     
       2. The apparatus of  claim 1 , wherein the microdroplet-manipulating device comprises a syringe fitted with a tip. 
     
     
       3. The apparatus of  claim 2 , wherein the controller is programmed to cause the syringe, whenever the microdroplet is in a chamber of the vessel and submerged in the hydrophobic liquid:
 to hold the microdroplet in a pendant manner from the tip and 
 to move the microdroplet relative to the hydrophobic liquid. 
 
     
     
       4. The apparatus of  claim 2 , wherein the microdroplet-manipulating device further comprises a vibration-producing device disposed in contact with the syringe so as to, when actuated, vibrate the syringe in an orbital manner sufficiently to impart a corresponding stirring motion of the microdroplet in contact with the tip. 
     
     
       5. The apparatus of  claim 1 , wherein the vessel comprises a first chamber containing a hydrophobic liquid held at a nucleic acid-denaturation temperature, a second chamber containing the hydrophobic liquid held at a nucleic acid-annealing temperature, and a third chamber containing the hydrophobic liquid held at a nucleic acid-extending temperature. 
     
     
       6. The apparatus of  claim 5 , wherein:
 the microdroplet-manipulating device comprises a syringe fitted with a tip; and 
 the controller is programmed to cause the syringe to retract the microdroplet into the tip whenever the microdroplet is being moved, by respective motion of the syringe imparted by the movement and placement device, through at least one of the plurality of channels from one chamber to the next. 
 
     
     
       7. The apparatus of  claim 1 , wherein the controller is programmed to execute at least two protocols on a microdroplet, the protocols including a first protocol comprising at least one step performed with the microdroplet on the superhydrophobic surface and a second protocol comprising at least one step performed with the microdroplet in the vessel submerged in the hydrophobic liquid. 
     
     
       8. The apparatus of  claim 7 , wherein the first protocol is directed to at least one of releasing genetic material from cells in the microdroplet, diluting the microdroplet, centrifuging the microdroplet, lysing the microdroplet, precipitating genetic material in the microdroplet, collecting genetic material from the microdroplet, washing and drying collected genetic material from the microdroplet, and rehydrating collected genetic material from the microdroplet. 
     
     
       9. The apparatus of  claim 8 , wherein:
 the microdroplet-manipulating device comprises a syringe fitted with a tip; 
 the first protocol is directed to at least collecting genetic material from the microdroplet; and 
 collecting genetic material from the microdroplet comprises adhering the genetic material to the tip. 
 
     
     
       10. The apparatus of  claim 9 , wherein collecting genetic material further comprises rotating the tip as the genetic material adheres to the tip. 
     
     
       11. The apparatus of  claim 5 , wherein the controller is programmed to command the movement and placement device to perform the recited acts of:
 (c) removing at least a portion of the microdroplet from the superhydrophobic surface, 
 (d) placing the microdroplet in a chamber of the temperature-controlled vessel, and 
 (e) manipulating the microdroplet in the chamber; and 
 wherein the controller is further programmed to command the movement and placement device to place the microdroplet-manipulating device proximate to the chambers of the vessel in a cyclic manner at respective times, each cycle comprising: 
 locating the microdroplet-manipulating device proximate to the first chamber of the vessel at which the microdroplet-manipulating device submerges a microdroplet being carried by the microdroplet-manipulating device in the hydrophobic liquid in the first chamber for a first defined length of time; 
 transporting the microdroplet through to the second chamber through a first one or the plurality of channels, while keeping the droplet submerged in the hydrophobic liquid; 
 holding the microdroplet in the hydrophobic liquid in the second chamber for a second defined length of time; 
 transporting the microdroplet to the third chamber through a second one of the plurality of channels, while keeping the droplet submerged in the hydrophobic liquid; 
 holding the microdroplet in the hydrophobic liquid in the third chamber for a third defined length of time; and 
 returning the microdroplet to the first chamber through a third one of the plurality of channels, while keeping the droplet submerged in the hydrophobic liquid; 
 wherein the cycle is commanded to be repeated a plurality of times. 
 
     
     
       12. The apparatus of  claim 5 , wherein the controller is programmed to command the movement and placement device to perform each of the recited acts of:
 (c) removing at least a portion of the microdroplet from the superhydrophobic surface, 
 (d) placing the microdroplet in a chamber of the temperature-controlled vessel, and 
 (e) manipulating the microdroplet in the chamber. 
 
     
     
       13. The apparatus of  claim 12 , wherein the controller is programmed to command the movement and placement device to place the microdroplet-manipulating device proximate to the chambers of the vessel in a cyclic manner at respective times, each cycle comprising:
 locating the microdroplet-manipulating device proximate to the first chamber of the vessel at which the microdroplet-manipulating device submerges a microdroplet being carried by the microdroplet-manipulating device in the hydrophobic liquid in the first chamber for a first defined length of time; 
 transporting the microdroplet through to the second chamber through a first one or the plurality of channels, while keeping the droplet submerged in the hydrophobic liquid; 
 holding the microdroplet in the hydrophobic liquid in the second chamber for a second defined length of time; 
 transporting the microdroplet to the third chamber through a second one of the plurality of channels, while keeping the droplet submerged in the hydrophobic liquid; 
 holding the microdroplet in the hydrophobic liquid in the third chamber for a third defined length of time; and 
 returning the microdroplet to the first chamber through a third one of the plurality of channels, while keeping the droplet submerged in the hydrophobic liquid; 
 wherein the cycle is commanded to be repeated a plurality of times. 
 
     
     
       14. The apparatus of  claim 5 , wherein the controller is programmed to command the movement and placement device to perform each of the recited acts of:
 (d) placing the microdroplet in the first chamber of the temperature-controlled vessel, 
 (e) manipulating the microdroplet in the first chamber, and 
 (f) removing at least a portion of the microdroplet from the first chamber. 
 
     
     
       15. A method, comprising:
 providing a movement and placement device defining a preset motion range in at least two of x-, y-, and z-dimensions; 
 providing a microdroplet-manipulating device coupled to and movable by the controlled movement and placement device in the preset range; 
 providing a superhydrophobic surface located in the preset range; 
 providing a temperature-controlled vessel located in the preset range, the vessel comprising:
 multiple interconnected chambers, each chamber comprising a respective volume of a hydrophobic liquid held at a respective temperature, and 
 a plurality of open-top channels interconnecting the chambers, each channel comprising a respective volume of the hydrophobic liquid; 
 
 providing a controller operably connected to the movement and placement device and to the microdroplet-manipulating device, the controller being programmed to:
 command the movement and positioning device to place the microdroplet-manipulating device relative to the superhydrophobic surface, and 
 command the movement and placement device to perform automatically at least one of:
 (a) placing an aqueous liquid microdroplet on the superhydrophobic surface, 
 (b) manipulating the microdroplet on the superhydrophobic surface, or 
 (c) removing at least a portion of the microdroplet from the superhydrophobic surface; and 
 
 at least one of:
 (d) placing the microdroplet in a first chamber of the temperature-controlled vessel, 
 (e) manipulating the microdroplet in the first chamber, or 
 (f) removing at least a portion of the microdroplet from the first chamber; 
 
 
 placing an amount of a sample in contact with the superhydrophobic surface using the microdroplet-manipulating device coupled to a movement and placement device defining a preset motion range in at least two of x-, y-, and z-dimensions to form at least one microdroplet of the sample on the superhydrophobic surface; and 
 performing at least one step of a first protocol on, in, or with the microdroplet on the superhydrophobic surface. 
 
     
     
       16. The method of  claim 15 , wherein the at least one step is selected from the group consisting of:
 moving the microdroplet on the superhydrophobic surface, 
 adding a substance to the microdroplet on the superhydrophobic surface, 
 removing a substance from the microdroplet on the superhydrophobic surface, 
 mixing contents of the microdroplet on the superhydrophobic surface, 
 concentrating the microdroplet on the superhydrophobic surface, 
 agitating the microdroplet on the superhydrophobic surface, 
 changing a volume of the microdroplet on the superhydrophobic surface, 
 changing a shape of the microdroplet on the superhydrophobic surface, 
 changing a density of the microdroplet on the superhydrophobic surface, 
 changing a composition of the microdroplet on the superhydrophobic surface, 
 changing a position of the microdroplet on the superhydrophobic surface, 
 holding a substance relative to the microdroplet on the superhydrophobic surface, 
 merging the microdroplet with another microdroplet on the superhydrophobic surface, 
 splitting the microdroplet on the superhydrophobic surface, and 
 rotating the microdroplet on the superhydrophobic surface. 
 
     
     
       17. The method of  claim 15 , further comprising:
 removing at least part of the microdroplet from the superhydrophobic surface using the microdroplet-manipulating device; 
 placing the removed at least part of the microdroplet in contact with a hydrophobic liquid, using the microdroplet-manipulating device, to form a second microdroplet in the hydrophobic liquid; and 
 performing at least one step of a second protocol on, in, or with the second microdroplet in the hydrophobic liquid. 
 
     
     
       18. The method of  claim 17 , wherein:
 the second protocol is a thermocycling protocol; and 
 the at least one step of the second protocol comprises keeping the second microdroplet immersed in the hydrophobic liquid at a specified temperature for a specified period of time. 
 
     
     
       19. The method of  claim 15 , wherein the microdroplet-manipulating device comprises a syringe fitted with a tip. 
     
     
       20. The method of  claim 19 , wherein the controller is programmed to cause the syringe, whenever the microdroplet is in a chamber of the vessel and submerged in the hydrophobic liquid:
 to hold the microdroplet in a pendant manner from the tip, and 
 to move the microdroplet relative to the hydrophobic liquid. 
 
     
     
       21. The method of  claim 19 , wherein the microdroplet-manipulating device further comprises a vibration-producing device disposed in contact with the syringe so as to, when actuated, vibrate the syringe in an orbital manner sufficiently to impart a corresponding stirring motion of the microdroplet in contact with the tip. 
     
     
       22. The method of  claim 15 , wherein the multiple interconnected chambers comprise a first chamber comprising a hydrophobic liquid held at a nucleic acid-denaturation temperature, a second chamber comprising the hydrophobic liquid held at a nucleic acid-annealing temperature, and a third chamber comprising the hydrophobic liquid held at a nucleic acid-extending temperature. 
     
     
       23. The method of  claim 22 , wherein:
 the microdroplet-manipulating device comprises a syringe fitted with a tip; and 
 the controller is programmed to cause the syringe to retract the microdroplet into the tip whenever the microdroplet is being moved, by respective motion of the syringe imparted by the movement and placement device, through at least one of the plurality of channels from one chamber to the next. 
 
     
     
       24. The method of  claim 15 , wherein the first protocol is directed to at least one of: releasing genetic material from cells in the microdroplet, diluting the microdroplet, centrifuging the microdroplet, lysing the microdroplet, precipitating genetic material in the microdroplet, collecting genetic material from the microdroplet, washing and drying collected genetic material from the microdroplet, or rehydrating collected genetic material from the microdroplet. 
     
     
       25. The method of  claim 24 , wherein:
 the microdroplet-manipulating device comprises a syringe fitted with a tip; and 
 the first protocol is directed to at least collecting genetic material from the microdroplet; and 
 collecting genetic material from the microdroplet comprises adhering the genetic material to the tip. 
 
     
     
       26. The method of  claim 25 , wherein the collecting the genetic material further comprises rotating the tip as the genetic material adheres to the tip.